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Abstract:

Described herein are antimicrobial compounds identified via a
high-throughput inhibitor screen of the in vitro activity of MipZ, which
is an ATPase that regulates division site placement in Caulobacter
crescentus. The compounds and their analogs are active against bacterial
membranes and thus represent a novel class of antimicrobial compounds.
The antimicrobial compounds are effective against both actively growing
bacterial cells as well as bacterial cells in the stationary phase. The
antimicrobial compounds are also effective against bacteria in biofilms.

Description:

[0003] The present disclosure is related to broad-spectrum antimicrobial
compounds, pharmaceutical compositions comprising the antimicrobial
compounds, and methods of treating bacterial infections with the
antimicrobial compounds.

BACKGROUND

[0004] While the prevalence of multi-drug resistant pathogens continues to
rise, the rate at which new clinical antimicrobials are introduced has
declined significantly. In addition, the treatment of persistent
infections has been complicated by pathogen phenotypes. Bacteria that
grow very slowly are often associated with prolonged infections, and they
are particularly tolerant to many of the clinically important classes of
antibiotics that inhibit rapidly growing cells. For example, the
β-lactam family of antibiotics inhibits enzymes involved in the
synthesis of peptidoglycan, and is thus most effective at targeting
microbes that grow rapidly and continuously synthesize new cell wall.
Relying on antibiotics that require fast metabolism and growth creates
long-term problems, as dormant bacteria, as well as those associated with
biofilms and other multicellular structures, may survive antibiotic
treatments, become predisposed to developing drug resistance, and cause a
relapse.

[0005] An effective strategy for combating slow-growing bacteria is to
target the lipid membrane. Proteomic analyses have demonstrated that
approximately one third of all proteins in bacteria are associated with
membranes. Peripheral and integral membrane proteins participate in
various essential cellular processes, including: nutrient and waste
transport, respiration, adhesion, mobility, cell-cell communication, and
the transfer of genetic material. Compounds that perturb these processes
disrupt growth and the maintenance of cell homeostasis and may serve as
potent therapeutic antimicrobial agents.

[0006] Synthetic and naturally occurring small molecules that disrupt the
bacterial membrane have been developed to treat persistent infections of
Mycobacterial and Staphylococcal species. This class of compounds
exhibits multiple mechanisms of action, including: the inhibition of
specific enzymatic processes in the membrane, decreasing the
trans-membrane potential (ΔΨ), and increasing membrane
permeability. The increase in permeability may act as a double-edged
sword, as it perturbs bacterial physiology and facilitates the
penetration of free radicals secreted by macrophages of the host immune
system.

[0007] The therapeutic benefit of membrane-active drugs has been
demonstrated against dormant bacteria; however, there are no clear design
rules for small molecules that are specific for bacterial versus
eukaryotic membranes. Many of the members of this class of antibiotics
are ineffective against Gram-negative bacteria, presumably due to the
outer membrane. The identification of new broad-spectrum antibiotics that
target bacterial membranes and the study of their mechanism of toxicity
would provide an important step forward for this field.

[0008] What are needed are new broad-spectrum antimicrobial compounds,
particularly antimicrobial compounds that target bacterial membranes.

BRIEF SUMMARY

[0009] In one aspect, included herein is an antimicrobial compound of
Formula I, or a pharmaceutically acceptable salt thereof:

[0017] R3 and R4 in --C(R2)(R3)(R4) together with
the carbon (--C) form a cycloalkyl or heterocycloalkyl ring structure.

[0018] In another aspect, included herein are pharmaceutical compositions
containing the compounds of formula I and methods of treating a subject
in need of treatment for a bacterial infection comprising administering
to the subject the pharmaceutical compositions.

[0019] In a further aspect, included herein are of methods inhibiting
bacterial growth comprising contacting bacteria with an antimicrobially
effective amount of a compound of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a schematic showing division plane assembly in
Caulobacter crescentus and the concentration gradient of MipZ, which
concentrates FtsZ at the division plane.

[0021] FIG. 2 is a schematic of the assay used to identify inhibitors of
MipZ.

[0022] FIG. 3 shows an analysis of MipZ-YFP localization. (A) Plot of
percent of population with wild type localization for treatment with
different compounds at different concentrations. (B) Representative
fluorescence images of C. crescentus cells expressing MipZ-YFP.

[0024] FIG. 5: (A, B) Measurement of ΔΨ using DiOC2 A) Data
for B. subtilis (n≦8,964 cells). B) Data for C. crescentus
(n≧158 cells). C) Measurement of B. subtilis membrane permeability
using PI (n≧2,546 cells). D) Measurement of C. crescentus membrane
permeability using PI (n≧35 cells). In box plots, the top whisker
represents 95%, the bottom whisker is 5%, the top of the box is 75%, the
bottom of the box is 25%, and the line inside the box indicates the
median of each sample population. Three asterisks (***) refers to
p<0.001.

[0027] FIG. 8 shows a rabbit red blood cell (RBC) hemolysis assay. Columns
represent average values, and error bars represent the standard deviation
of the mean for three replicates. Assay performed using the MIC
conditions for C. crescentus and S. aureus (A), the MSC for C. crescentus
(B), and the MSC for S. aureus (C).

[0028] The above-described and other features will be appreciated and
understood by those skilled in the art from the following detailed
description, drawings, and appended claims.

DETAILED DESCRIPTION

[0029] Described herein is a family of compounds that specifically target
the membranes of both Gram-positive and Gram-negative bacteria. In a
specific embodiment, described herein is the compound,
(2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxym-
ethyl)propane-1,3-diol) (referred to as DCAP), and its derivatives. DCAP
was identified via a high-throughput inhibitor screen of the in vitro
activity of MipZ, which is an ATPase that regulates division site
placement in Caulobacter crescentus. Using a strain of C. crescentus in
which MipZ was translationally fused to yellow fluorescent protein (YFP),
treatment of cells with DCAP (20 μM) caused MipZ-YFP to mislocalize.
At high concentrations of DCAP (≧75 μM), cell lysis was
observed within minutes after treatment. Without being held to theory, it
is believed that DCAP does not specifically inhibit MipZ in the cell, but
instead alters the properties of the cell envelope.

[0030] The chemical structure of DCAP is given below. The stereogenic
center is marked with an *.

##STR00001##

[0031] In one embodiment, an antimicrobial compound has the Formula I, or
a pharmaceutically acceptable salt thereof:

[0047] R3 and R4 in --C(R2)(R3)(R4) together with
the carbon (--C) form a C3-C7 cycloalkyl.

[0048] Further within this embodiment, R1 is hydrogen, methyl, or
ethyl.

[0049] Still further within this embodiment, R2, R3, and R4
are each independently hydrogen, methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, pentyl, isopentyl, --CH2OH, --CH2SH,
--CH2CH2OH, --CH2CH2SH, --OCH3,
--OCH2CH3, --CH2OCH3, --CH2SCH3, or R2
is hydrogen and R3 and R4 in --C(R2)(R3)(R4)
together with the carbon (--C) form a cyclohexyl.

[0050] In another embodiment, the inhibitor includes compounds and
pharmaceutically acceptable salts of Formula Ia to Ih:

[0059] In certain situations, the compounds of Formulae I, Ia, and Ib may
possess asymmetry so that the compounds can exist in different
stereoisomeric forms. These compounds can be, for example, racemates or
optically active forms. For compounds with two or more asymmetric
elements, these compounds can additionally be mixtures of diastereomers.
For compounds having asymmetric centers, it should be understood that all
of the optical isomers and mixtures thereof are encompassed. In addition,
compounds with carbon-carbon double bonds may occur in Z- and E-forms,
with all isomeric forms of the compounds being included in the present
invention. In these situations, the single enantiomers, i.e., optically
active forms, can be obtained by asymmetric synthesis, synthesis from
optically pure precursors, or by resolution of the racemates. Resolution
of the racemates can also be accomplished, for example, by conventional
methods such as crystallization in the presence of a resolving agent, or
chromatography, using, for example a chiral LC or HPLC column.

[0060] The term "substituted", as used herein, means that any one or more
hydrogens on the designated atom or group is replaced with a selection
from the indicated group, provided that the designated atom's normal
valence is not exceeded. When a substituent is oxo (i.e., ═O), then 2
hydrogens on the atom are replaced. When aromatic moieties are
substituted by an oxo group, the aromatic ring is replaced by the
corresponding partially unsaturated ring. For example a pyridyl group
substituted by oxo is a pyridone. Combinations of substituents and/or
variables are permissible only if such combinations result in stable
compounds or useful synthetic intermediates. A stable compound or stable
structure is meant to imply a compound that is sufficiently robust to
survive isolation from a reaction mixture, and subsequent formulation
into an effective therapeutic agent.

[0061] A dash ("--") that is not between two letters or symbols is used to
indicate a point of attachment for a substituent. For example, --COOH is
attached through the carbon atom.

[0062] As used herein, "alkyl" is intended to include both branched and
straight-chain saturated aliphatic hydrocarbon groups, having the
specified number of carbon atoms. Thus, the term C1-C8 alkyl as
used herein includes alkyl groups having from 1 to about 8 carbon atoms.
When C0-Cn alkyl is used herein in conjunction with another
group, for example, phenylC0-C4alkyl, the indicated group, in
this case phenyl, is either directly bound by a single covalent bond
(C0), or attached by an alkyl chain having the specified number of
carbon atoms, in this case from 1 to about 2 carbon atoms. Examples of
alkyl include, but are not limited to, methyl, ethyl, n-propyl,
isopropyl, n-butyl, t-butyl, n-pentyl, and sec-pentyl.

[0063] "Alkenyl" as used herein, indicates hydrocarbon chains of either a
straight or branched configuration comprising one or more unsaturated
carbon-carbon bonds, which may occur in any stable point along the chain,
such as ethenyl and propenyl.

[0064] "Alkynyl" as used herein, indicates hydrocarbon chains of either a
straight or branched configuration comprising one or more triple
carbon-carbon bonds that may occur in any stable point along the chain,
such as ethynyl and propynyl.

[0066] "Alkanoyl" indicates an alkyl group as defined above, attached
through a keto (--(C═O)--) bridge. Alkanoyl groups have the indicated
number of carbon atoms, with the carbon of the keto group being included
in the numbered carbon atoms. For example a C2alkanoyl group is an
acetyl group having the formula CH3(C═O)--.

[0067] The term "alkoxycarbonyl" indicates an alkoxy group, as defined
above, having the indicated number of carbon atoms, attached through a
keto linkage. The carbon of the keto linker is not included in the
numbering, thus a C2alkoxycarbonyl has the formula
CH3CH2O(C═O)--.

[0068] The term "alkylcarboxamide" indicates an alkyl group, as defined
above, having the indicated number of carbon atoms, attached through a
carboxamide linkage, i.e., a --CONH2 linkage, where one or both of
the amino hydrogen is replace by an alkyl group. Alkylcarboxamide groups
may be mono- or di-alkylcarboxamide groups, such an ethylcarboxamide or
dimethylcarboxamide.

[0069] As used herein, the term "mono- or di-alkylamino" indicates
secondary or tertiary alkyl amino groups, wherein the alkyl groups are as
defined above and have the indicated number of carbon atoms. The point of
attachment of the alkylamino group is on the nitrogen. Examples of mono-
and di-alkylamino groups include ethylamino, dimethylamino, and
methyl-propyl-amino.

[0070] As used herein, the term "aryl" indicates aromatic groups
containing only carbon in the aromatic ring or rings. Such aromatic
groups may be further substituted with carbon or non-carbon atoms or
groups. Typical aryl groups contain 1 to 3 separate, fused, or pendant
rings and from 6 to about 18 ring atoms, without heteroatoms as ring
members. Where indicated aryl groups may be substituted. Such
substitution may include fusion to a 5 to 7-membered saturated cyclic
group that optionally contains 1 or 2 heteroatoms independently chosen
from N, O, and S, to form, for example, a 3,4-methylenedioxy-phenyl
group. Aryl groups include, for example, phenyl, naphthyl, including
1-naphthyl and 2-naphthyl, and bi-phenyl.

[0071] In the term "(aryl)alkyl", aryl and alkyl are as defined above, and
the point of attachment is on the alkyl group. This term encompasses, but
is not limited to, benzyl, phenylethyl, and piperonyl. Likewise, in the
term (aryl)carbhydryl, aryl and carbhydryl are as defined above and the
point of attachment is on the carbhydryl group, for example a
phenylpropen-1-yl group.

[0072] "Carbhydryl" as used herein, includes both branched and
straight-chain hydrocarbon groups, which are saturated or unsaturated,
having the specified number of carbon atoms.

[0073] "Cycloalkyl" as used herein, indicates saturated hydrocarbon ring
groups, having the specified number of carbon atoms, usually from 3 to
about 8 ring carbon atoms, or from 3 to about 7 carbon atoms. Examples of
cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or
cyclohexyl as well as bridged or caged saturated ring groups such as
norbornane or adamantane.

[0074] "Haloalkyl" indicates both branched and straight-chain saturated
aliphatic hydrocarbon groups having the specified number of carbon atoms,
substituted with 1 or more halogen atoms, generally up to the maximum
allowable number of halogen atoms. Examples of haloalkyl include, but are
not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and
penta-fluoroethyl.

[0075] "Haloalkoxy" indicates a haloalkyl group as defined above attached
through an oxygen bridge.

[0076] "Halo" or "halogen" as used herein refers to fluoro, chloro, bromo,
or iodo.

[0077] As used herein, "heteroaryl" indicates a stable 5- to 7-membered
monocyclic, 7-to 10-membered bicyclic, or 10- to 15-membered heterocyclic
ring which contains at least 1 aromatic ring that contains from 1 to 4,
or preferably from 1 to 3, heteroatoms chosen from N, O, and S, with
remaining ring atoms being carbon. When the total number of S and O atoms
in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to
one another. It is preferred that the total number of S and O atoms in
the heteroaryl group is not more than 2. Examples of heteroaryl groups
include, but are not limited to, pyridyl, indolyl, pyrimidinyl,
pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl,
thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl,
pyrazolyl, and 5,6,7,8-tetrahydroisoquinoline. In the term
"heteroarylalkyl," heteroaryl and alkyl are as defined above, and the
point of attachment is on the alkyl group. This term encompasses, but is
not limited to, pyridylmethyl, thiophenylmethyl, and pyrrolyl(1-ethyl).

[0078] The term "heterocycloalkyl" is used to indicate saturated cyclic
groups containing from 1 to about 5 heteroatoms chosen from N, O, and S,
with remaining ring atoms being carbon. Heterocycloalkyl groups have from
3 to about 8 ring atoms, and more typically have from 5 to 7 ring atoms.
A C2-C7heterocycloalkyl group contains from 2 to about 7 carbon
ring atoms and at least one ring atom chosen from N, O, and S. Examples
of heterocycloalkyl groups include morpholinyl, piperazinyl, piperidinyl,
and pyrrolidinyl groups.

[0079] "Pharmaceutically acceptable salts" includes derivatives of the
disclosed compounds wherein the parent compound is modified by making an
acid or base salt thereof, and further refers to pharmaceutically
acceptable solvates of such compounds and such salts. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines; alkali or
organic salts of acidic residues such as carboxylic acids; and the like.
The pharmaceutically acceptable salts include the conventional salts and
the quaternary ammonium salts of the parent compound formed, for example,
from inorganic or organic acids. For example, conventional acid salts
include those derived from inorganic acids such as hydrochloric,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the
salts prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,
maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,
mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,
toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,
HOOC--(CH2)n--COOH where n is 0-4, and the like. The
pharmaceutically acceptable salts of the present invention can be
synthesized from a parent compound that contains a basic or acidic moiety
by conventional chemical methods. Generally, such salts can be prepared
by reacting free acid forms of these compounds with a stoichiometric
amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide,
carbonate, bicarbonate, or the like), or by reacting free base forms of
these compounds with a stoichiometric amount of the appropriate acid.
Such reactions are typically carried out in water or in an organic
solvent, or in a mixture of the two. Generally, non-aqueous media like
ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are
preferred, where practicable.

[0080] In one aspect, provided herein are methods of treating a subject in
need of treatment for a bacterial infection, comprising administering to
the individual an antimicrobial compound or composition as described
herein. The bacteria can be actively growing or in the stationary phase.

[0082] In another aspect, a method of inhibiting bacterial growth
comprises contacting the bacteria with an antimicrobial compound as
described herein. The bacteria can be actively growing or in the
stationary phase. Methods of inhibiting bacteria include methods useful
for treatment of a subject (human or veterinary) and also include methods
useful for inhibiting bacteria outside of a subject, such as use for
sterilization and disinfection.

[0083] In one embodiment, the bacteria are in the form of a biofilm. A
biofilm is a complex aggregate of microorganisms such as bacteria,
wherein the cells adhere to each other on a surface. The cells in
biofilms are physiologically distinct from planktonic cells of the same
organism, which are single cells that can float or swim in liquid medium.
Biofilms are involved in, for example, urinary tract infections, middle
ear infections, dental plaques, gingivitis, coatings of contact lenses,
cystic fibrosis, and infections of joint prostheses and heart valves.

[0084] The antimicrobial compounds and compositions may be administered
prophylactically, chronically, or acutely. For example, such compounds
may be administered prophylactically to patients known to be prone to
bacterial infections, or who are known to have been exposed to
potentially infectious agents. The compounds may also be administered
prophylactically to patients suffering other conditions, such as AIDS or
other immune-system-suppressing conditions that render them susceptible
to opportunistic infections. In addition to the prevention of such
infections, chronic administration of the antimicrobial compounds will
typically be indicated in treating refractory conditions, such as
persistent infection by multiple drug-resistant strains of bacteria.
Acute administration of the antimicrobial compounds is indicated to
treat, for example, those subjects presenting with classical indications
of bacterial infection.

[0085] As used herein, "contacting" means that a compound is provided such
that it is capable of making physical contact with another element, such
as a microorganism, a microbial culture or a substrate. In another
embodiment, the term "contacting" means that the compound is introduced
into a subject receiving treatment, and the compound is allowed to come
in contact in vivo. Thus, contacting can include administration of a
compound, that is, introducing the compound into the body, such as into
the systemic circulation. Administration routes include but are not
limited to, rectal, oral, buccal, sublingual, pulmonary, transdermal,
transmucosal, as well as subcutaneous, intraperitoneal, intravenous, and
intramuscular injection.

[0086] Since the antimicrobial compounds are antibacterially active and
inhibit bacterial growth, they are also of use in treating bacterial
contamination of a substrate, such as hospital instruments or work
surfaces. In order to treat a contaminated substrate, the compounds may
be applied to the site of such contamination in an amount sufficient to
inhibit bacterial growth.

[0087] In certain embodiments, the compounds are administered to a patient
or subject. A "patient" or "subject", used equivalently herein, means
mammals and non-mammals. "Mammals" means a member of the class Mammalia
including, but not limited to, humans, non-human primates such as
chimpanzees and other apes and monkey species; farm animals such as
cattle, horses, sheep, goats, and swine; domestic animals such as
rabbits, dogs, and cats; laboratory animals including rodents, such as
rats, mice, and guinea pigs; and the like. Examples of non-mammals
include, but are not limited to, birds, and the like. The term "subject"
does not denote a particular age or sex.

[0088] The phrase "effective amount," as used herein, means an amount of
an agent, which is sufficient enough to significantly and positively
modify symptoms and/or conditions to be treated (e.g., provide a positive
clinical response). The effective amount of an active ingredient for use
in a pharmaceutical composition will vary with the particular condition
being treated, the severity of the condition, the duration of the
treatment, the nature of concurrent therapy, the particular active
ingredient(s) being employed, the particular pharmaceutically-acceptable
excipient(s)/carrier(s) utilized, and like factors within the knowledge
and expertise of the attending physician. In general, the use of the
minimum dosage that is sufficient to provide effective therapy is
preferred. Patients may generally be monitored for therapeutic
effectiveness using assays suitable for the condition being treated or
prevented, which will be familiar to those of ordinary skill in the art.

[0089] The phrase "inhibitory amount", as used herein, means an amount of
an agent (a compound or composition), which is sufficient to reduce the
level or activity of bacterial infection to a statistically significant
lesser value as compared to when the agent is not present.

[0090] The amount of compound effective for any indicated condition will,
of course, vary with the individual subject being treated and is
ultimately at the discretion of the medical or veterinary practitioner.
The factors to be considered include the condition being treated, the
route of administration, the nature of the formulation, the subject's
body weight, surface area, age and general condition, and the particular
compound to be administered. In general, a suitable effective dose is in
the range of about 0.1 to about 500 mg/kg body weight per day, preferably
in the range of about 5 to about 350 mg/kg per day. The total daily dose
may be given as a single dose, multiple doses, e.g., two to six times per
day, or by intravenous infusion for a selected duration. Dosages above or
below the range cited above may be administered to the individual patient
if desired and necessary.

[0091] Also included herein are pharmaceutical compositions comprising the
antimicrobial compounds. As used herein, "pharmaceutical composition"
means a therapeutically effective amount of the compound together with a
pharmaceutically acceptable excipient, such as a diluent, preservative,
solubilizer, emulsifier, adjuvant, and the like. As used herein
"pharmaceutically acceptable excipients" are well known to those skilled
in the art.

[0092] Tablets and capsules for oral administration may be in unit dose
form, and may contain excipients such as binding agents, for example
syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone;
fillers for example lactose, sugar, maize-starch, calcium phosphate,
sorbitol or glycine; tabletting lubricant, for example magnesium
stearate, talc, polyethylene glycol or silica; disintegrants for example
potato starch, or acceptable wetting agents such as sodium lauryl
sulphate. The tablets may be coated according to methods well known in
normal pharmaceutical practice. Oral liquid preparations may be in the
form of, for example, aqueous or oily suspensions, solutions, emulsions,
syrups or elixirs, or may be presented as a dry product for
reconstitution with water or other suitable vehicle before use. Such
liquid preparations may contain conventional additives such as suspending
agents, for example sorbitol, syrup, methyl cellulose, glucose syrup,
gelatin hydrogenated edible fats; emulsifying agents, for example
lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may
include edible oils), for example almond oil, fractionated coconut oil,
oily esters such as glycerine, propylene glycol, or ethyl alcohol;
preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic
acid, and if desired conventional flavoring or coloring agents.

[0093] For topical application to the skin, the drug may be made up into a
cream, lotion or ointment. Cream or ointment formulations that may be
used for the drug are conventional formulations well known in the art.
Topical administration includes transdermal formulations such as patches.

[0094] For topical application to the eye, the inhibitor may be made up
into a solution or suspension in a suitable sterile aqueous or
non-aqueous vehicle. Additives, for instance buffers such as sodium
metabisulphite or disodium edeate; preservatives including bactericidal
and fungicidal agents such as phenyl mercuric acetate or nitrate,
benzalkonium chloride or chlorhexidine, and thickening agents such as
hypromellose may also be included.

[0095] The active ingredient may also be administered parenterally in a
sterile medium, either subcutaneously, or intravenously, or
intramuscularly, or intrasternally, or by infusion techniques, in the
form of sterile injectable aqueous or oleaginous suspensions. Depending
on the vehicle and concentration used, the drug can either be suspended
or dissolved in the vehicle. Advantageously, adjuvants such as a local
anaesthetic, preservative and buffering agents can be dissolved in the
vehicle.

[0096] Pharmaceutical compositions may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in the
art of pharmacy. The term "unit dosage" or "unit dose" means a
predetermined amount of the active ingredient sufficient to be effective
for treating an indicated activity or condition. Making each type of
pharmaceutical composition includes the step of bringing the active
compound into association with a carrier and one or more optional
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing the active compound into association
with a liquid or solid carrier and then, if necessary, shaping the
product into the desired unit dosage form.

[0097] The antimicrobial compounds may also be administered in combination
with an additional active agent, such as, for example, an inhibitor of
bacterial efflux. Efflux pumps are proteins that unidirectionally remove
antibiotics from cytoplasmic compartments, and are considered to be a
mechanism of antibacterial resistance. Bacterial efflux inhibitors
include chalcone compounds as disclosed in WO 11/075,136, the polybasic
compounds disclosed in WO 10/054,102, the quaternary alkyl ammonium
functional compounds disclosed in WO 08/141,012, the compounds disclosed
in WO 05/007162, the substituted polyamines of WO 04/062674, which are
incorporated herein by reference in their entirety.

[0101] In vivo screen with a Caulobacter crescentus strain that expresses
MipZ-YFP. Hits from in vitro screens were tested for activity in vivo. A
C. crescentus strain (MT97) that expresses mipZ-yfp from the native mipZ
promoter was used. An overnight culture of MT97 was diluted to an
absorbance (2=600 nm) of ˜0.1; the diluted culture was grown
further for at least 1 hour prior to compound treatment. Compounds were
mixed with a solution of 1% agarose in M2G media to achieve a final
concentration of 20 μM. The cells (1 μL per pad) were transferred
on top of the compound-containing agarose pad, and the cell morphology
and localization of MipZ-YFP were imaged for a period of 24 hours using
epifluorescence microscopy. In between microscopic observations, the
inoculated pads were incubated at 30° C. to promote growth.

[0102] Effects of compounds on MipZ localization. A strain of C.
crescentus in which MipZ was translationally fused to yellow fluorescent
protein (YFP) was used to determine the effect of DCAP on MipZ
localization. For experiments with C. crescentus expressing MipZ-YFP,
overnight cultures were diluted 10-fold and incubated at least 1 hour
prior to treatment with compounds. After adding compounds, cells were
incubated at 200 rpm and 30° C. for 20 minutes. Cells were imaged
as described above. Following cell segmentation and fluorescence signal
calculation in MicrobeTracker, a separate MATLAB script was used to
detect signal peaks within an individual cell. The number of total peaks
and peak locations were calculated (i.e., poles or mid-cell) for each
cell. Poles were defined as 1-25% and 75-100% along the normalized cell
length (1-100%). Cells were classified as `wildtype` if their catalogued
information agreed with one of the following criteria: 1) MipZ-YFP was
unipolar, meaning there was one peak and the peak resides within a pole
region; and 2) MipZ-YFP was bipolar, meaning there were two peaks and
both peaks are within a polar region. After this classification, a
contingency table was created with the total number of cells analyzed,
and the number of cells with the `wildtype` localization. Using the
GraphPad InStat program, the Fisher's exact test was applied to calculate
two-sided p-values between DMSO and compound-treated cells.

[0103] Fluorescence microscopy and flow cytometry to measure ΔΨ.
The fluorescent probe, 3,3'-diethyloxacarbocyanine iodide (DiOC2)
was used to measure ΔΨ in DMSO and in compound-treated cells.
To eliminate the possibility of an interaction between the probe and
compounds, the fluorescence intensity of solutions of DiOC2 was
measured in the presence and absence of the compounds as described in the
art. To measure ΔΨ in B. subtilis cells, flow cytometry was
used. Cell suspensions were labeled with 30 μM DiOC2 and filtered
through a Nylon filter containing 60-μm diameter pores. The
fluorescence of these cells was measured using a BD LSR II flow
cytometer. The following instrument settings were used for detection: low
flow rate, 488 nm excitation laser, 530/30 nm emission filter for green
fluorescence, and 575/26 nm emission filter for red fluorescence. FlowJo
software was used to analyze flow cytometry data. First, a gate was drawn
around the region where cells were detected in the forward versus side
scatter area plot. Gating enabled the exclusion of any non-cellular
materials in the sample that the instrument detected. The ratio of
red-to-green fluorescence was calculated for each particle within the
gate using the software, and the resulting data was exported to GraphPad
Prism to create box plots shown in FIG. 5. Statistical parameters were
calculated using the Kruskal-Wallis non-parametric one-way analysis of
variation (GraphPad InStat).

[0104] For experiments with C. crescentus cells, the cells were prepared
by diluting the overnight culture 10-fold into fresh PYE medium. The
diluted culture was grown for 1 hour at 200 rpm and 30° C. The
cells were treated with compounds for 10 minutes at room temperature,
DiOC2 dye was added to a final concentration of 30 μM, and incubated
for 10 minutes. After labeling, small aliquots (1-2 μl) of the
suspensions of cells were transferred onto 1% agarose pads for
fluorescence microscopy. A Nikon Eclipse TE2000E inverted microscope with
an Andor DU-895 EMCCD camera, a Perfect Focus system, and an encoded
z-stage were used for phase contrast and epifluorescence microscopy. For
detecting green and red fluorescence of the dye, the following
wavelengths (λex/λem): 484/520 nm and 555/620 nm,
respectively, were used. Acquired images were analyzed using the
MATLAB-based script MicrobeTracker. Using this software, the area of
individual cells in the phase contrast images was segmented. The
segmentation was applied to the fluorescence images to calculate the
fluorescence intensity for individual cells. A separate MATLAB script was
written to process the results from MicrobeTracker, in which the
cell-area normalized ratio of red to green fluorescence for each cell was
calculated. This data was exported to GraphPad Prism and GraphPad InStat
for creating box plots and performing statistical analysis (one-way
analysis of variation), respectively.

[0105] Measurement of membrane permeability using fluorescence microscopy
and flow cytometry. The DNA-binding probe, propidium iodide (PI) was used
to measure the relative membrane permeability between DMSO, ethanol (50%
v/v for C. crescentus and 70% v/v for B. subtilis) and compound-treated
cells. To eliminate the possibility of an interaction between the
fluorophore and small molecules, the fluorescence intensity of solutions
of PI was measured in the presence and absence of the compounds. For
experiments with B. subtilis cells, flow cytometry was used as described
in the previous section, except the fluorescence settings in the
instrument were as follows: 488 nm excitation laser, 610/20 nm emission
filter. When using FlowJo to analyze the data, the auto-fluorescence from
cells was excluded from PI fluorescence. Other details for the analysis
are identical to the description in the previous section.

[0106] For experiments with C. crescentus cells, the cells were prepared
by diluting the overnight culture 10-fold into fresh PYE medium. The
diluted culture was grown for 1-3 hours at 200 rpm and 30° C. The
cells were treated with compounds for 10 minutes at room temperature, PI
was added at a final concentration of 20 μM, and incubated for 10
minutes. Image acquisition and data analysis were performed as described
in the previous section.

[0107] Measurement of compound effects on FtsA: For experiments with C.
crescentus expressing Venus-FtsA, a synchronized population of cells was
used. First, an overnight culture was grown with kanamycin (5 μg/mL)
and glucose (0.02% w/v). The presence of glucose suppressed the
transcription of venus-ftsA from its xylose-inducible promoter. 2 mL of
this culture was diluted into 25 mL of fresh PYE with the antibiotic and
glucose, and incubated further to achieve an absorbance of ˜0.6
(λ=600 nm). Once the cells reached mid-exponential phase, the
culture was centrifuged for 10 minutes at 6000 rpm and 4° C. The
cell pellet was resuspended in ice-cold M2 (a final volume of
approximately 1000 μL) and 750 μL of this suspension was added to
an equal-volume of sterile Percol (Sigma Aldrich). After thoroughly
mixing the solution, it was centrifuged for 20 minutes at 11,000 rpm and
4° C. Upon centrifugation, the bottom band (swarmer cells) was
taken and the cells were washed in ice-chilled M2 solution (1 mL). The
washed cell pellet was suspended in fresh PYE containing kanamycin (no
glucose, 6 mL). The cell suspension was incubated for 20 minutes and then
xylose (0.03% w/v) was added to induce the expression of venus-ftsA.
Cells were grown for another 20 minutes and subsequently treated with
compounds. The cells were incubated for 20 minutes again in the presence
of the compounds prior to imaging. Thus, the total time of xylose
induction was 40 minutes, and the total time of compound treatment was 20
minutes. At this time point (60 minutes post synchrony), the majority of
the cells were at the beginning of cell division. Imaging conditions and
data analysis were identical to the conditions described for MipZ-YFP,
except for a set of criteria used for defining `wildtype` protein
localization. `Wildtype` was defined as 1) Venus-FtsA is unipolar,
meaning there was one peak, and the peak resided within a polar region;
and 2) Venus-FtsA was at the mid-cell, meaning there was one peak and it
was within the mid-cell region (40-60% of the normalized cell length).

[0108] Measurement of compound effects on MinD: B. subtilis DS4294
(amyE::Pxyl-GFP-minD cat) cells were grown to exponential phase
(λ=600 nm, 0.4-0.7) in LB with incubation at 30° C. and 200
rpm shaking. Cells were induced for GFP-MinD production by adding xylose
to the media to a final concentration of 0.1% (w/v) followed by
incubating for 75 min. After induction, cells were treated with compounds
for 20 minutes before imaging. Other details for imaging conditions and
data analysis were identical to those described for MipZ-YFP, except for
the definition of poles, and a set of criteria used for defining
`wildtype` protein localization. `Wildtype` was defined as 1) GFP-MinD
was bipolar, meaning there were two peaks, and both peaks were at the
poles (1-20% and 80-100% of the normalized cell length); 2) GFP-MinD was
at the mid-cell in addition to the bipolar localization, meaning there
were three peaks with two of them at the poles and one at mid-cell; and
3) GFP-MinD was at an quaternary position (20-40% and 60-80% of the
normalized cell length) in addition to the bipolar and mid-cell
localization (the total number of peaks was 4 if there is one quaternary
peak, and the total was 5 if there are two quaternary peaks). Because B.
subtilis cells can initiate division prior to finishing an earlier
division and the completion of septation, these normal cells have peaks
at quaternary positions. Any cells that did not fit these criteria were
categorized as non-wildtype.

[0109] Determination of the minimum inhibitory concentration (MIC) of
bacterial growth. The MIC of various organisms in liquid media was
determined using the macrodilution method according to the NCCLS
guidelines. For the starting inoculum, cultures were diluted to contain
approximately 5×105 cells/mL in growth media. To create a
two-fold dilution series for the macrodilution technique, each compound
was added to the first culture tube (4 mL total volume) at the highest
concentration. 2 mL of this culture was diluted into an equal volume of
inoculated media (a two-fold dilution). The final volume for each culture
was 2 mL. The MIC was determined by identifying the lowest concentration
of compound that completely inhibited growth by visual inspection.

[0111] Cultures of C. crescentus (24 hour incubation) and S. aureus cells
(5 day incubation) were grown from single colonies. Cells were collected
by centrifugation (8500 rpm for 2.5 minutes) and re-suspended in M2 salt
solution (for C. crescentus; 1.74 g/L Na2HPO4, 1.06 g/L
KH2PO4, and 0.5 g/L NH4Cl) or phosphate-buffered saline
(for S. aureus; PBS, Fisher Scientific). The centrifugation and
re-suspension was repeated two more times. After the repeated washing
steps, the cell suspension was diluted 10-fold to achieve approximately
108 cells/mL. Aliquots of the diluted cell suspension were
transferred into wells of a 96-well plate (100 μL/well), and 2-fold
serial dilutions were performed to test a range of antibiotic
concentrations. The plate was sealed with Parafilm® and incubated for
24 hours at room temperature in the dark. After incubation, 50 μl was
removed from each well to spread on nutrient agar plates (1.5% agar in
PYE for C. crescentus, and LB for S. aureus). The colonies on each plate
were counted after growing the cells for 1-2 days. For determining the
kinetics of bactericidal activity of DCAP on stationary cultures, the
washed cells were diluted by 100-fold in appropriate solutions and tested
using a single concentration of the compound while including a DMSO
control sample. Cell suspensions were kept in closed microcentrifuge
tubes instead of the wells of 96-well plates. 100 μL of the
suspensions was removed for plating at each time point. All MSC
experiments were performed in static conditions.

[0112] Determination of the minimum biofilm inhibitory concentration of
growth (bMIC) and biofilm eradication concentration (MBEC). Overnight
cultures of C. crescentus and S. aureus were grown and diluted 100-fold
into appropriate nutrient media. Aliquots of the diluted suspension were
transferred into wells of 96-well plates (150 μL/well). 2-fold serial
dilutions were performed for compound-containing wells. For every
experiment, three replicates for each compound concentration tested were
included. After transferring aliquots of the suspension, the plates were
closed with specialized lids that have protruding pins (Nunc STP System),
and the plates were sealed using Parafilm®. The plates were incubated
in a static incubator at 30° C. (C. crescentus) or 37° C.
(S. aureus) for 24 hours. After the cells had formed biofilms on the
surface of the pins, the pins were rinsed by dipping them in M2 (C.
crescentus) or PBS (S. aureus) solutions. To rinse the pins, clean,
sterile 96-well plates were used (aliquots of 200 μL/well); each rinse
lasted 10 sec. After washing away planktonic cells loosely bound to
biofilms, the pins were inserted into a 96-well plate that was
pre-aliquoted with nutrient media containing antibiotics (2-fold
dilutions, a final volume of 150 μL/well). The plates were sealed with
Parafilm®, and incubated for 17 hours, and the bMIC was measured at
the end of the incubation by visual inspection. After the bMIC run, the
rinse steps were repeated to remove planktonic cells, and the pins were
inserted into a 96-well plate that was pre-aliquoted with drug-free
nutrient media (aliquots of 150 μL/well). The plates were sealed with
Parafilm®, incubated for 24 hours, and the MBEC was measured at the
end of the incubation by visual inspection.

[0113] Rabbit red blood cell (RBC) hemolysis assay. Rabbit RBCs from
Lampire Biological Laboratories were used. Prior to preparing the RBCs,
compound-containing PBS solutions were serially diluted into a 96-well
plate (a final volume of 100 μL/well). The RBC lysis solution
(Epicentre Biotechnology) was included as a positive control for
hemolysis. For each assay, 1 mL of the RBC suspension was removed from
the stock bottle and centrifuged for 2 minutes at 2000 rpm. The pelleted
cells were resuspended in sterile PBS solution and centrifuged again. The
cells were resuspended in PBS and diluted 5-fold into the same solution.
100 μL aliquots of RBCs were added into wells of a 96-well plate that
contained an equal volume of a solution of compound in PBS. The plates
were incubated for varying amounts of time depending on the microbial
assay conditions to be emulated. For MIC-like conditions, plates were
incubated for at least 17 hours at 30° C. or 37° C. For
MSC-like conditions, the incubation time was either 2 hours or 6 hours at
30° C. or 37° C. During the incubation, un-lysed RBCs
settled at the bottom of the wells. At the end of the incubation, 90
μL of the supernatant was transferred into the wells of a fresh
96-well plate, and the absorbance of the heme at 2=405 nm was measured.

Example 1

Selection of Compounds

[0114] In Caulobacter crescentus cells, the mechanism of accurately
placing FtsZ in time and space in the cell involves an ATPase, MipZ.
(FIG. 1) The ATP-bound form of MipZ promotes depolymerization of FtsZ
filaments by increasing the GTP hydrolysis rate of FtsZ. In addition to
its inhibition of FtsZ, MipZ binds to nucleoproteins near the origin of
the chromosome and establishes an asymmetric polar gradient. The gradient
becomes bipolar (i.e., symmetric) when the cell undergoes chromosome
replication to place two origins at opposite poles. This bipolar gradient
of MipZ positions the division at the mid-cell: FtsZ is localized at the
mid-cell, where the concentration of the inhibitory gradient of MipZ is
the lowest. Thus, MipZ coordinates chromosome segregation and the onset
of cell division both spatially and temporally such that FtsZ assembles
at the mid-cell after the segregation of the chromosomes. Because of the
relationship between FtsZ and MipZ, a coupling assay and a fluorescence
polarization assay of MipZ ATPase activity were used to screen compounds
for their ability to inhibit cell division. (FIG. 2)

[0115] In vitro ATPase screen with purified recombinant MipZ. Recombinant
MipZ for in vitro screens was prepared as described in the art
(Thanbichler, M.; Shapiro, L. Cell 2006, 126, 147.). Two ATPase assays
were used to screen three small molecule libraries (a total of 43,400
compounds) at the University of Wisconsin Carbone Cancer Center. One
assay utilized pyruvate kinase and lactic dehydrogenase as coupling
enzymes, and phosphoenolpyruvate and NADH as their substrates,
respectively. A solution of coupling enzymes, their substrates, Triton
X-100 and MipZ were aliquoted (22.3 μL, per well) in 384-well black
plates using Biomek FX liquid handler (Beckman Colter). Plates were
briefly centrifuged to pull liquids to the bottom of the wells. Pin tools
were used to deliver 0.2 μL, of a stock solution of unique small
molecules (10 mM in DMSO) from chemical libraries to each well. The first
two columns of each plate were reserved for controls and did not receive
compounds from the libraries. A solution of ATP (2.5 μL, per well) was
added using a Biotek Fill instrument to all wells except the first column
for each plate to initiate ATP hydrolysis. Final concentrations of assay
components were: 0.01% Triton X-100, 1 mM phosphoenolpyruvate, 0.3 mM
NADH, 3 U/mL pyruvate kinase, 3 U/mL lactic dehydrogenase, 7.5 μM
MipZ, and 1 mM ATP in a buffered solution of 50 mM Tris-HCl, 50 mM KCl,
and 10 mM MgCl2. The plates were gently vortexed to mix the solution
and incubated for 3 hours at 30° C. After incubation, the
fluorescence emission from NADH was measured using a Tecan Safire II®
plate reader (λex=340/35 nm; λem=460/10 nm).
Measurements of the fluorescence intensity from control wells were used
to calculate the Z-factor; the minimum Z-factor for all plates was 0.7.
The coupling enzyme assay was used to screen compounds from Maybridge and
Life Chemicals libraries. Compounds that inhibited ≧60% of ATP
hydrolysis compared to the positive control were identified as hits and
were screened using a secondary assay to eliminate compounds that target
coupling enzymes. The secondary assay consisted of the same reaction
components as the primary assay, except for the omission of MipZ and ATP.
A solution of ADP was added instead. The fluorescence emission of
compounds that did not inhibit coupling enzymes was measured at the
specified wavelengths used for NADH, and the compounds were retested for
their inhibitory effect on MipZ activity in vitro.

[0116] In addition to the coupling enzyme assay, a fluorescence
polarization (FP) assay was used to monitor the ATPase activity of MipZ
in vitro. Reaction conditions and component concentrations were the same
as the coupling enzyme assay unless noted otherwise. The FP assay
utilized anti-ADP antibodies and Alexa633-labeled ADP. A
Transcreener® ADP2 FP assay kit from BellBrook Labs (Madison,
Wis.) was used. A solution of MipZ was aliquoted (10 μL per well) into
plates, and addition of ATP (1 μL of 5 mM stock solution per well)
initiated the reaction. After 3 hours, 10 μL of ADP detection mix (541
μg/mL of antibody) was added to each well, and the plates were
incubated for 1 hour at 25° C. The following wavelengths were used
for FP measurements: 635 nm for excitation and 670/20 nm for emission.
The Z-factor for the FP assay was ≧0.7. The FP assay was used for
screening compounds from the Life Chemicals library and the Spectrum
Collection. Hits from the FP assay were tested for inhibition of the
anti-ADP antibody by repeating the assay in the absence of MipZ, and
checked for their intrinsic fluorescence at the specified wavelengths
used for the Alexa633 probe.

[0117] Hits were further screened for antibacterial activity using a
Caulobacter crecentus strain that expresses MipZ-YFP as described in
Materials and Methods. DCAP was confirmed as having antibacterial
activity in the MipZ-YFP assay (data not shown).

[0118] (2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hy-
droxymethyl)propane-1,3-diol) (DCAP) was identified in the screen.

Example 2

Confirmation of the DCAP Structure

[0119] The identification of
(2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxym-
ethyl)propane-1,3-diol) (DCAP) was confirmed by nuclear magnetic resonance
(NMR) spectroscopy and high resolution electrospray ionization. DCAP used
in all experiments was purchased from Ryan Scientific (catalog number
F3255-0148, Mt. Pleasant, S.C.). A Varian MercuryPlus 300 MHz instrument
(Magnetic Resonance Facility in the Chemistry Department of the
University of Wisconsin-Madison) was used to obtain 1D 1H and
13C NMR spectra for DCAP at 25° C. A Bruker Avance III 500
MHz instrument (National Magnetic Resonance Facility at Madison) was used
to collect 2D 13C-HMBC, 15N-HMBC, HSQC, COSY, and TOCSY spectra
for DCAP structure verification. The solvent used was deuterated dimethyl
sulfoxide. The data were analyzed using Sparky (T. D. Goddard and D. G.
Kneller, University of California, San Francisco) and NUTS (Acorn NMR).
The NMR data is summarized below:

[0122] DCAP was also analyzed using high resolution electrospray
ionization mass spectrometry in positive ion mode to determine the exact
mass of the compound (Mass Spectrometry Facility, Department of Chemistry
at the University of Wisconsin-Madison). Expected masses were
(M+H)+=413.0952 and (M+Na)+=435.0849. Measured mass was
435.0832 (Data not shown).

Example 3

Determination of MipZ Localization in C. Crescentus in the Presence of
DCAP

[0123] Using a strain of C. crescentus in which MipZ was translationally
fused to yellow fluorescent protein (YFP), it was found that the
treatment of cells with DCAP (20 μM) caused MipZ-YFP to mislocalize
(FIG. 3). As a positive control, carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) was used. In FIG. 3, `wildtype` (WT) localization was
defined as 1) unipolar (a single peak of fluorescence signal at a pole)
and 2) bipolar (two peaks at poles). All images were acquired 20 min
after treating cells with compounds. At least 69 cells were analyzed for
each sample. Two-sided p-values using Fisher's exact test in comparison
to DMSO: p=0.0075 (**) for 25 μM of CCCP, p=0.0002 (***) for 20 μM
of DCAP, and p<0.0001 (***) for 100 μM of DCAP.

[0124] At high concentrations of DCAP (≧75 μM), cell lysis
within minutes after treatment was observed (FIG. 4). This observation
suggested to us that DCAP may not specifically inhibit MipZ in the cell
but instead alter the properties of the cell envelope.

Example 4

Effect of DCAP on Membrane Potential

[0125] To test the hypothesis that DCAP alters the properties of the cell
envelope, the membrane potential in the presence and absence of DCAP was
measured.

[0126] ΔΨ was measured for two model bacteria, C. crescentus
(Gram-negative) and Bacillus subtilis (Gram-positive), in the absence and
presence of DCAP. As a positive control, carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) was used. CCCP increases the permeability of protons
across the membrane and decreases ΔΨ. To visualize changes in
the membrane potential, the fluorescent probe 3,3'-diethyloxacarbocyanine
iodide (DiOC2) was used. DiOC2 emits green fluorescence (λ=530
nm) in its monomeric form. Its fluorescence emission maximum is
red-shifted (λ=576 nm) upon self-association. A large ΔΨ
stimulates the aggregation of DiOC2 and produces a high ratio of
λ576/λ530. Conversely,
λ576/λ530 decreases when ΔΨ is dissipated
in bacteria. FIGS. 5A and B illustrate λ576/λ530
for cells treated with DMSO, CCCP, and DCAP. A significant decrease in
λ576/λ530 was apparent after 20 minutes of
treatment with CCCP and DCAP (p<0.001) and indicated that the
ΔΨ was dissipated rapidly. Antibiotics that do not target the
bacterial membrane can decrease the potential over a long period of
exposure (e.g. 3-4 hours); however, the rapid action of DCAP suggests
that the dissipation in ΔΨ was due to its direct effect on the
membrane.

Example 5

Effect of DCAP on Membrane Permeability

[0127] The mechanism of action of DCAP was explored. One possibility is
that DCAP functions as an ionophore similar to CCCP. Alternatively, DCAP
may increase the general permeability of the membrane. To investigate the
mechanism, propidium iodide (PI) was used to label the DNA of cells with
compromised membranes. As shown in FIGS. 5C and D, ethanol-treated cells
were intensely labeled with PI, while the DMSO control sample was not.
Treating cells with CCCP did not increase DNA labeling with PI; the
fluorescence emission of these cells was similar to the DMSO sample.
Addition of DCAP to cells increased the fluorescence of cells labeled
with PI, although the intensity was significantly lower than the
ethanol-treated cells (P<0.001). These results suggest that DCAP has
at least two mechanisms of antimicrobial action: it decreases
ΔΨ by facilitating ion transport across the membrane and has a
minor effect on the general permeability of the lipid bilayer.

Example 6

Effect of DCAP on In Vivo Localization of Division Proteins MinD and FtsA

[0128] ΔΨ was recently identified as an important parameter for
the in vivo localization of division proteins associated with the
bacterial membrane, including MinD and FtsA. The treatment of B. subtilis
cells with either CCCP or DCAP altered the localization of a fusion of
green fluorescent protein to MinD (GFP-MinD) compared to DMSO-treated
cells (FIG. 6A). MinD localizes to the poles of B. subtilis cells and
guides division plane formation at the mid-cell. As division progresses,
MinD accumulates at the mid-cell and marks the sites of future cell
poles. Treating B. subtilis cells expressing GFP-MinD with CCCP led to
more diffuse fluorescence throughout the cell but had little effect on
the location of the signal compared to the DMSO control (FIG. 7A). In
DCAP-treated cells, GFP-MinD mislocalized--the number of fluorescent foci
increased in some cells while in others the fluorescence signal became
more diffuse and was no longer concentrated at the poles (FIG. 7A).

[0129] In addition to perturbing the localization of MinD in B. subtilis,
CCCP and DCAP influenced the distribution of FtsA in C. crescentus. FtsA
is a peripheral membrane protein that interacts with FtsZ and activates
the recruitment of downstream division proteins. FtsA resides at one pole
(i.e., the pole opposite to the stalk) in non-dividing C. crescentus
cells and is recruited to the mid-cell as division begins. To study cells
at this stage of division, cells that express a fluorescent fusion of the
protein, Venus-FtsA, were synchronized and then treated with CCCP and
DCAP. Treatment of C. crescentus cells with CCCP at its minimum
inhibitory concentration (5 μM) did not alter the localization of
FtsA; however, a higher concentration of CCCP (25 μM) had a
significant effect on the position of FtsA. Most cells treated with DCAP
exhibited multiple peaks of FtsA fluorescence (≧2) instead of a
single peak--either at the pole or mid-cell--observed in untreated cells
(FIG. 7B). The observation that DCAP causes the mislocalization of
membrane proteins in B. subtilis and C. crescentus is consistent with the
hypothesis that the compound decreases ΔΨ. The effect of DCAP
was similar to the ionophore CCCP; however, DCAP causes more severe
protein mislocalization at its MIC, which may arise from its influence on
membrane permeability.

Example 7

Measurement of Minimum Inhibitory Concentrations of DCAP

[0130] After confirming the membrane-targeting activity of DCAP, the
efficacy of DCAP against other bacterial species was tested. Table 2
demonstrates that DCAP inhibits the growth of Escherichia coli,
Pseudomonas aeruginosa, and Staphylococcus aureus. Deleting one or more
efflux pumps in E. coli and P. aeruginosa strains increased the
sensitivity of cells to DCAP. Efflux pumps are active against a broad
range of compounds and typically consist of three components: two
transmembrane proteins, one in the inner and the other in the outer
membrane, and a periplasmic protein that connects the two transmembrane
components. Deleting to/C whose product is embedded in the outer membrane
in E. coli strain BW25113 reduced the MIC of DCAP by 4-fold. This result
suggests that the activity of DCAP in Gram-negative bacteria is largely
due to its interaction with the inner membrane.

[0131] In addition to its activity against actively growing bacteria, DCAP
kills cells in stationary phase (Table 3). This property was tested
against C. crescentus and S. aureus. S. aureus was used as a model
Gram-positive bacterium rather than B. subtilis for these experiments, as
B. subtilis can sporulate under starvation conditions and does not form
robust biofilms on the plastic surfaces used as substrates. To ensure
that bacteria were deprived of nutrients, the cells were grown into late
stationary phase (i.e., 24 hours for C. crescentus and 5 days for S.
aureus). These cells were collected, suspended in isotonic solutions
devoid of amino acids or sugars, and treated with a small molecule (DMSO,
DCAP, CCCP or ampicillin). Cell viability was measured over time by
plating culture aliquots on non-selective, solid growth media. The
minimum concentration of antibiotic required to completely eliminate
colony formation was designated as the minimum stationary-bactericidal
concentration (MSC). The MSC and MIC of DCAP for each organism were
similar while the efficacy of ampicillin was significantly reduced for
stationary cells of both organisms (Table 3). The MSC of ampicillin for
S. aureus was 1000-fold higher than its MIC, while the MSC of ampicillin
for C. crescentus was beyond the range of the measurements. CCCP
inhibited the proliferation of C. crescentus cells regardless of their
physiological status. However, the MSC of CCCP for S. aureus was
>300-fold higher than the MIC. This dramatic decrease in the
effectiveness of CCCP in S. aureus may be due to changes in membrane
composition as cells adjust their metabolism in nutrient-deprived
conditions. Overall, CCCP and DCAP were more effective in killing
stationary cells than ampicillin.

[0132] Membrane-active compounds are also efficient at eradicating
biofilm-associated cells (Table 2). Biofilms are implicated in a wide
range of human diseases including cystic fibrosis and urinary tract
infection and are particularly recalcitrant to antibiotics. The
heterogeneity in the physiology of cells in biofilms makes it possible
for the bacterial communities to persist in stressful conditions. To
determine the efficacy of antibiotics against biofilms, the minimum
biofilm inhibitory concentration (bMIC) and the minimum biofilm
eradication concentration (MBEC) were measured. Briefly, cell suspensions
from overnight cultures were diluted and transferred into the wells of a
96-well plate; the lid of the plate contained 96 individual plastic pins
that protruded into each of the 96 wells. Biofilms formed on the surface
of the pins after incubation for 24 hours and were transferred to growth
media and serially dosed with DCAP, CCCP, or ampicillin. After 24 hours
of exposure to compounds, the lowest concentration of antibiotic that
inhibited planktonic cell growth in the wells (bMIC) was determined.
Since bMIC is a measurement of the rapid growth of freely suspended cells
released from biofilms in the presence of antibiotics, the bMIC and MIC
values did not differ significantly (Table 2).

[0133] After performing bMIC experiments, biofilms growing on the pins of
the lid were transferred to nutrient media devoid of antibiotics to
measure the minimum concentration of antibiotic that prevented planktonic
growth from biofilms in antibiotic-free nutrient media (MBEC). MBEC
indicates whether the exposure to antimicrobial agent used during the
bMIC experiment sterilized biofilm-associated cells. MBEC values were
generally larger than MICs and indicated an increased tolerance of stress
exhibited by cells associated with biofilms. For C. crescentus, the MBEC
values recapitulated the trend observed in the MSC: CCCP and DCAP
effectively eradicated biofilm cells while ampicillin was not cytotoxic
at the highest concentration tested (400 μM). CCCP was the only
effective antibiotic against S. aureus biofilms. Since CCCP was not as
effective as DCAP at killing stationary cells, it was hypothesized that
this variability in efficacy of membrane-active drugs is in part caused
by changes in membrane composition (i.e., membrane proteins and lipid
content) at different developmental stages of bacterial cells. Despite
the variations in efficacy, it was concluded that the comparison of MIC,
bMIC, MSC, and MBEC measurements for the three antibiotics support the
hypothesis that membrane-active drugs eradicate dormant, slow-growing
bacteria more effectively than antibiotics that rely on growth-dependent
mechanisms.

Example 8

Toxicity of DCAP in Mammalian Cells

[0134] To test the toxicity of DCAP against mammalian membranes, the
hemolysis of rabbit red blood cells (RBC) was measured. These experiments
were performed using conditions that closely mimicked the MIC and MSC
assays. RBCs were treated with CCCP and DCAP at their MICs for the time
periods used (17 hours) to determine the MIC of C. crescentus and S.
aureus. After incubation, the absorbance of heme released from lysed RBCs
was measured. The MIC concentrations of CCCP and DCAP did not
significantly disrupt RBC membranes (FIG. 8A) although higher
concentrations of DCAP (i.e., 50 μM) were moderately toxic to RBCs.

[0135] The MSC assay conditions were also reproduced to measure toxicity
against RBCs. First, the minimum time required to obtain the MSC for DCAP
treatment of C. crescentus (2 hours) and S. aureus (6 hours; data not
shown) was determined. Using these times, the hemolysis assay was
performed and no significant toxicity of DCAP against RBCs was observed
(FIGS. 8B and C). In contrast, CCCP was toxic to RBCs at a high
concentration (FIG. 8C). These measurements indicate that DCAP does not
appreciably perturb mammalian membranes in conditions that are lethal to
C. crescentus and S. aureus.

[0136] In summary, the discovery and characterization of the
membrane-active antimicrobial agent DCAP and its analogs has been
reported. DCAP kills bacteria by depolarizing ΔΨ and increasing
membrane permeability. These activities disrupt the organization and
integrity of the bacterial membrane and mislocalize essential,
membrane-associated proteins (e.g. MinD and FtsA). DCAP is inert to
mammalian membranes at concentrations at which it is a potent
antibacterial agent. Analogs of DCAP may yield more potent antimicrobial
agents. Future studies with these compounds may provide insight into
changes in the properties of membranes during the life cycle of bacteria
and their correlation to the vulnerability of cells to membrane-active
drugs.

[0137] Finally, studies of the structure-function relationship of DCAP and
other broad-spectrum compounds may provide design rules for potent
membrane-targeting drugs that kill bacterial cells specifically.

Example 9

Experimental Protocol for the General Synthesis of DCAP

##STR00047##

[0138] Scheme 1: General Synthetic Route for the Synthesis of DCAP Analogs

Synthetic Step 1: N-alkylation

[0139] In a dry round bottom flask, 3,6-dichlorocarbazole (705 mg, 3.0
mmol) was added to a solution of KOH (1.2 equiv., 3.6 mmol) in DMF (20
mL) at 0° C. After stirring the mixture for 30 min at 4°
C., (+/-)-epichlorohydrin (2.5 equiv., 7.2 mmol) was added to the cooled
flask drop wise. The reaction was stirred at 0° C. overnight. The
reaction was removed from the ice bath and allowed to warm to room
temperature. H2O (20 mL) was added to the mixture and white solid
precipitant formed. The heterogeneous mixture was filtered and washed
with H2O three times to provide a solid consisting of a mixture of
N-alkylated product and remaining starting material. After applying high
vacuum overnight, the white solid was used directly in any epoxide
opening reaction.

Synthetic Step 2: Epoxide Opening

[0140] In a dry round bottom flask, the white solid from step 1 (100 mg,
0.344 mmol) was dissolved into ethanol (4 mL) and an amine (2 equiv.,
0.68 mmol) was added to the mixture. The reaction was refluxed overnight
under argon using an oil bath at 78° C. After cooling to room
temperature, the ethanol was evaporated. The mixture was purified by
flash column chromatography on silica gel with 5-20%
methanol/dichloromethane to provide a DCAP analog.

Example 10

MIC Data for DCAP Analogs

[0141] Several analogs of DCAP have been synthesized and their activity
against E. coli and other bacteria have been tested.

[0142] Bacterial Strains and Growth Conditions:

[0143] Luria-Bertani (LB) media (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast
extract, pH 7.0) was used to grow all the strains represented in Tables 5
and 6. All the cultures were grown at 37° C. All the E. coli
strains were grown while shaking at 200 rpm and the clinical pathogens
were statically incubated.

[0145] The MIC of E. coli strains was determined in liquid LB media in
culture tubes (1 mL/tube) using the macrodilution method according to the
CLSI guidelines as is known in the art. For clinical pathogenic organisms
including S. typhimurium, V. cholera, S. boydii, K pneumonia, E.
aerogenes, A. baumannii, E. tarda, and M. morganii strains, liquid LB
media in 96-well microplates (100 μL/well) and the microdilution
method from the CLSI guidelines were used.

[0146] The structure-activity relation studies provide insight into the
regions of DCAP that modulate its biological activity. Modifications to
the carbazole, heterocyclic ring reduced the MIC of the analogs, while
changes to the tris-hydroxymethyl groups increased activity.
Specifically, replacing the tris-hydroxymethyl groups with substituents
that increased the hydrophobicity of the analog and decreased the MIC.
These general rules provide a guide to the activity of additional
analogs.

[0147] The use of the terms "a" and "an" and "the" and similar referents
(especially in the context of the following claims) are to be construed
to cover both the singular and the plural, unless otherwise indicated
herein or clearly contradicted by context. The terms first, second etc.
as used herein are not meant to denote any particular ordering, but
simply for convenience to denote a plurality of, for example, layers. The
terms "comprising", "having", "including", and "containing" are to be
construed as open-ended terms (i.e., meaning "including, but not limited
to") unless otherwise noted. Recitation of ranges of values are merely
intended to serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the specification as
if it were individually recited herein. The endpoints of all ranges are
included within the range and independently combinable. All methods
described herein can be performed in a suitable order unless otherwise
indicated herein or otherwise clearly contradicted by context. The use of
any and all examples, or exemplary language (e.g., "such as"), is
intended merely to better illustrate the invention and does not pose a
limitation on the scope of the invention unless otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element as essential to the practice of the invention as used
herein.

[0148] While the invention has been described with reference to an
exemplary embodiment, it will be understood by those skilled in the art
that various changes may be made and equivalents may be substituted for
elements thereof without departing from the scope of the invention. In
addition, many modifications may be made to adapt a particular situation
or material to the teachings of the invention without departing from the
essential scope thereof. Therefore, it is intended that the invention not
be limited to the particular embodiment disclosed as the best mode
contemplated for carrying out this invention, but that the invention will
include all embodiments falling within the scope of the appended claims.
Any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.

Patent applications by Douglas Benjamin Weibel, Madison, WI US

Patent applications by Katherine Ann Hurley, Madison, WI US

Patent applications in class Tricyclo ring system having the five-membered hetero ring as one of the cyclos

Patent applications in all subclasses Tricyclo ring system having the five-membered hetero ring as one of the cyclos